Steroids



United States Patent Ofitice 2,992,972 Patented July 18, 1961 STEROIDSHerbert C. Murray, Hickory Corners, and Durey H. Peterson, Kalamazoo,Mich., assignors to The Upjohn Company, Kalamazoo, Mich., a corporationof Michigan No Drawing. Original application Apr. 10, 1952, Ser. No.281,656. Divided and this application Mar. 3, 1958, Ser. No. 718,483

13 Claims. (Cl. 195-51) This invention relates to a novel process forthe production and use of esters ofl1,17a,21-trihydroxy-4-pregnene-3,20-dione.

This application is a division of application Serial No. 281,656, filedApril 10, 1952, now US. Patent 2,861,088, which is in turn acontinuation-in-part of application Serial No. 272,944, filed February23, 1952, now US. Patent 2,602,769, which is in turn acontinuation-in-part of application Serial No. 180,496, filed August 19,1950, now abandoned.

In the process of the present invention,1lu,l7oc,2l-trihydroxy-4-pregnene 3,20 dione and 11fi,17a,21trihydroxy-4-pregnene-3,20-dione are prepared from l70c,21dihydroxy-4-pregnene-3,20-di0ne by microbiological oxygenation, andesterified to produce 21-acyloxy11a,17udihydroxy-4-pregnene-3,20-diones,11a,21-diacyloxy-17othydroxy-4-pregnene-3,20diones, and21-acyloxy-11fi,17a-

dihydroxy4-pregnene-3,20-diones. The esters thus obtained are useful inthat they either have pharmacological activity per se or in that theycan be converted by oxidation of the ll hydroxy group to thecorresponding 21- acyloxy-17a-hydroxy-4-pregnene-3,1 1,20-trione.

The microbiological oxygenation is carried out with the employment of anoxygenating fungus which is a species of a genus of a family of theorder Mucorales or the oxygenating enzymes obtainable therefrom. Amongthe several families of this order, the genera of the familiesMucoraceae and Choanephoraceae are of greatest utility, and, among thegenera of these families of fungi, the Rhizopus, Cunninghamella, and'Mucor have been found of greatest value in the method of the presentinvention. Species of these genera which are operative in the method ofthe invention include, for example, the Rhizopus species microsporus,circinans, oligosporus, arrhizus, cohnii, oryzae, nigricans, chinensis,japonicus, tritici, kazensis, delemar, shanghaiensis, reflexus, andsynonymous species, which are in fact identical with those namedalthough differently named, and the representative Mucor species mucedo,griseo cyanus, hiemalis, hiemalis var. albus, rouxii, adventitius,christianiensis, circinelloides, dubius, genevensis, javanicus,microsporus, parasticus, gravensis, plumbeus, plumbeus var. spinesens,micro sporus, ramannianus, racemosus, and the like. While species ofgenera of families of the order Mucorales, and especially those of thefamily Mucoraceae, are broadly operative in the method of the presentinvention as the oxidizing fungi, or reasons of economy andproductivity, species of the genera Rhizopus and Cunninghamella arepreferably utilized for production of optimum yields of11,17,21-trihydroxy-4-pregnene-3,20-dione, although in certain casesunder particular circumstances the other genera and other species may bemost advantageously employed. Species of these mentioned genera,however, in particular demonstrate pronounced steroid oxygenatingactivity, and are thereby able, according to the method of theinvention, when suitably grown and contacted withl7a,21-dihydroxy-4-pregnene-3,20-dione, to accomplish oxygenation of theeleven position of the steroid on an industrially important scale.

Though species of certain genera of the fungi, of the genus Rhizopus inparticular, the genus Cunninghamella,

and the genus Mucor, all of which are of the families Mucoraceae andChoanephoraceae are practical for most abundant production of oxygenatedsteroids, and while economic considerations may confine preferredoperation to certain species and strains possessing a distinctivephysiological specificity, species of other families and genera of theorder Mucorales are also operative for the production of oxygenatedsteroids in industrially significant quantities.

For example, other useful representative genera of the Mucoraceaefamily, and representative species thereof, as identified in H. Zycha,Kryptogamenfiora der Mark Brandenburg, Band VIa, 1-264 (1934) includeParasitella (P. simplex), Zygorhynchus (Z. heterogamus, Z, moelleri),Circinella (C. spinosa), Actinomucor (A. repens), Pirella (P.circinans), Absidia (A. reflexa, A. glauca), Spinellus (S.sphaerosporus), Phycomyces (Ph. blakesleeanus, Ph. theobromatus),Sporodinia (Sp. grandis), Pilaira (P. a nomala), Pillobolus (P.crystallinus), Dicoccum (D. asperum), Tieghemella (T. orchidities, T.italica). Other Mucorales families with their genera and representativespecies include Thamnidiaceae: Thamnidium,(Th. elegans), Dicranophora(D. fulva), Chaetostylum (C. Fresenii), Helicostylurn (H. piriforme),Chaetocladium (Ch. Brefeldii); Choanephoraceae: Blakeslea (B. trispora),Choanephora (Ch. cucurbitarum),

Rhopalomyces (Rh. elegans), Cunninghamella (C. elegans, C. vertichllata), Thamnocophalis (Th. quadrupedata), Mycotypha (M.microspora); Cepalidaceae: Piptocephalis (P. preseniana), Syncephalis(S. reflexa, S. nodosa), Spinalia (Sp. radians), Syncephalastrum (S.racemosum, S. fulvium), Dispira (D. cornuta), Coomensia (C. pectinata),Kickxella (K. alabastrina); Mortierellaceae: Mortierella (M. pusilla, M.alpina), Haplosporangium (H. bisporale), Dissophora (D. decumbens);Endogenaceae: Endogone (E. reniformis), Sclerocystis (S. coromiodes),Glaziella (G. vesicztlosa).

Of the Rhizopus genus, species of which, as stated previously, arepreferred in the method of the invention, according to H. Zycha, Krypt.der Mark Brandenburg, Band VIa, -120 (1935), many commonly known speciesare synonymous. Thus Rhizopus microsporus may be known as Rh. minimus,Mucor speciosus, Rh. speciosus, and Rh. equinus; Rhizopus circinas maybe known as Rh. reflexus; Rhizopus oligosporus may be known as Rh.Delmar or Rh. Tamari; RhiZOpllS arrhizus may be known as Rh. nodosus,Rh. ramozus, Rh. maya'is, Mucor arrhizus, Mucor norvegicus, Rh.pusillus, Rh. bovinus, Rh. cambodja, Rh. chinensis, and Rh. tritici;Rhizopus cohnii may be known as Rh. Suinus; Rhizopus oryzae may be knownas Rh. japonicus or Rh. tonkinensis;

Rhizopus nigricans may be known as Mucor stolonifer,

Rh. niger, Rh. artocarpi, Mucor niger, Rh. nigricans var. minor, or Th.nigricans var. luxurians; and Rhizopus echinatus is itself a doubtfulspecies which may be synonymous with Rhizopus nigricans.

Of the Cunninghamella genus, the species, C. echinulata, C. bainieri, C.blakesleeana (ATCC 9245), C. eleans, C. verticulata, and C.berthollitiae, among others, have been used. These Cunninghamellaspecies and the Mucor javanicus, Mucor advantitious auranticus, Mucormicroporus (ATCC 8541) also demonstrate, in accordance with ourprocesses, the oxygenation of 17a,2l-dihydroxy-4-pregnene-3,20-dione.

The selected species of fungus of the order Mucorales is suitably grownon a medium containing available carbon, illustratively carbohydratessuch assugars or starches; assimilable nitrogen, illustratively solubleor insoluble proteins, peptones or amino acids; and mineralconstituents, illustratively phosphates and magnesium sulfate; and otherart recognized, desirable or adventitious, additions. The

'4' medium may desirably have a pH before innoculation of between about4.5 and 5.9 although a higher or lower pH may be used.

Inoculation of the fungal growth-supporting medium with the selectedfungus of the Mucorales order may be accomplished in any suitablemanner. Growth of the fungus is readily promoted by maintainingincubation temperatures of about room temperature, e.g. twenty totwenty-eight degrees centigrade, but a relatively wide range oftemperatures is suitable.

The period of fungal growth required before the 1711,21-dihydroxy-4-pregnene-3,20-dione is exposed to the oxygenatingactivity of the fungus does not appear to be critical. For example, thel7a,2l-dihydroxy-4-pregnene- 3,20-dione can be added either beforesterilization of the medium, at the time of inoculating the medium withthe selected Murcorales species, or at some time, e.g. 24 to 48 hours,later. The l7u,2l-dihydroxy-4-pregnene-3,20- dione to be oxygenated maybe added at any desired concentration although for practical reasonssteroid substrate at a concentration of about 0.01 gram to 0.25 gram orup to about 0.6 gram per liter of media or even 0.8 gram per liter issatisfactory although higher concentrations may be used with someinhibition of oxygenating activity. The addition of steroid substrate tobe oxygenated may be accomplished in any suitable manner so as topromote intimate contact of the steroid substrate with the oxygenatingactivity of the fungus and/ or fungal enzymes, such as by dispersing thesteroid substrate alone, with a dispersing agent, or in water-miscibleorganic solvent solution by mixing or homogenizing with the fungalmedium to form a suspension or dispersion of fine steroid particles.Either submerged or surface culture procedures may be employed withfacility, although submerged culture is preferred. Alternatively, theoxygenating activity or oxygenating enzymes of a growth of the fungusmay be separated from the fungus or the medium, admixed with the steroidor a solution or suspension thereof, and the mixture subjected toaerobic conditions to accomplish oxygenation of the steroid.

The temperature employed during the period of oxygenation of the steroidneed be maintained only within such ranges as support life, activegrowth or the oxygenating activity of the fungal organism.

While any form of aerobic incubation is satisfactory for the growth ofthe selected fungus or the bio-oxygenation of the steroid substrate, theefiiciency of oxygenation is related'to aeration. Therefore, controlledaeration, as by agitation and/ or blowing air through the fermentationmedium is usually employed.

The time required for the bio-oxygenation of the steroid variesaccording to the results desired. When the steroid substrate is presentat the time of inoculation of the medium, periods of from eight to 72hours may be used. However, when the steroid is added to the fungus,fungal medium, fungal enzymes, or the fermentation liquor aftersubstantial aerobic growth of the fungal organism, e.g. after 16 to 24hours, the conversion of the steroid substrate begins immediately andhigh yields are obtained in from one to 72 hours, 24 hours beingsatisfactory. In lieu of direct contact of the steroid to be oxygenatedwith the fermenting media containing viable Mucorales fungi, the steroidmay be oxygenated by contact with the fermented liquor, free of fungi,or with the fungi or enzymes of Mucorales preferably in the presence ofan aqueous menstruum, or with the enzymes obtained from the fungi.

After completion of the oxygenating fermentation reaction, the resultingl l,17a,2l-trihydroxy-4-pregnene-3 ,20 dione is recovered from thefermentation reaction mixture. An especially advantageous manner ofrecovering the l l,l7a,2l -trihydroxy-4-pregnene-3,20-dione involvesextracting the fermentation reaction mixture, including the fermentationliquor and. mycelia. in cases where the steroid is added directly to theculture, with a waterimmiscible organic'solvent, e.g. methylenechloride, ether, amyl acetate, and the like. The fermentation liquor andmycelia may be separated and separately extracted with suitablesolvents. The combined extracts are desirably washed with mild alkalinesolutions, and water, dried over anhydrous sodium sulfate, freed ofsolvent and purified by recrystallization from organic solvents or bychromatography.

The thus-produced l1,17a,21-trihydroxy-4-pregnene-3, ZO-dione crudefermentation product free of aqueous phase contaminants may be eitherpurified and separated or directly, and without prior purification orseparation, reacted with an acylating agent such as, for example,ketene, a ketene of a selected acid, an acid, acid chloride, acidanhydride, or other known acylating agent, usually in a solvent,illustratively benzene, toluene, or ether, and maintained at atemperature between about zero degrees centigrade and the boiling pointof the reaction mixture, suitably at about room temperature, for aperiod of time between about one-half hour and about hours. The time ofreaction as well as the temperature at which the reaction is performed,the acylating agent, and the ratio of reactants may be varied. Thereaction mixture may then be cooled or diluted by mixing with ice orcold water, and the product collected in an appropriate solvent which isthereafter washed with successive portions of a mildly basic solutionand water to obtain a solution of the product which is essentiallyneutral. After drying the solvent, the acylated11,17a,2l-trihydroxy-4-pregnene-3,20-dione may be isolated byevaporation of the solvent, and the residual product may be eitherdirectly oxidized to 21- acyloxy-l7a-hydroxy-4-pregnene-3,l1,20-trioneor it may be first purified by conventional procedures, such as, forexample, by recrystallization or chromatographic purification.

The thus-described acylationprocess, as illustrated in greater detail inthe examples following in this specification, produces both themono-esters and the di-esters, although in diiferent proportionsdepending upon the proportions of acylating agent toll,l7a,2l-trihydroxy-4- pregnene-3,20-dione. Using approximately oneequivalent of acylating agent to said steroid produces predominantly themono-acylated product, Whereas with about two or more equivalents ofacylating agent to said steroid, the di-acylated product has a greatertendency to form.

Either the crude or purified2l-acyloxy-1l,17a-dihydroxy-4-pregnene-3,ZO-dione may be oxidized withan oxidizing agent, illustratively chromium trioxide, to a 21- acyloxyester of cortisone (2l-acyloxy-l7a-hydroxy-4- pregnene-3,l1,20-trione)the production of which is an additional object of the present inventionand is shown in the examples.

The following preparations and examples are illustrative of the processand products of the present invention and are not to be construed aslimiting.

PREPARATION 1 11 ot,17a,21-trihydr0xy-4-pregnene-3,ZO-dione rate ofaeration and stirring such that the oxygen uptake was 6.3 to 7millimoles per liter per hour .of Na SO- ac cording to the method ofCooper, Fernstrom and Miller, Ind. Eng. Chem., 36, 504 (1944).

strain was added two grams of 17a,21-dihydroxy'-4-pregnene-3,20-dione(Reichsteins Substance S) in. 50 milli- 1 liters of acetone to provide asuspension of the steroid in To this medium containing a 24 hour growthof Rhizopus nigricalzs minus the culture. After an additional 96 hourperiod of incubation under the same conditions of temperature andaeration, the" beer and mycelium were extracted. The mycelium wasfiltered, washed twice, each time with a volume of acetone approximatelyequal to the volume of the mycelium and extracted twice, each time witha volume of methylene chloride approximately equal to the volume of themycelium. The acetone and methylene chloride extracts including solventwere added to the beer filtrate. The mixed extracts and beer filtratewere extracted successively with two one-half by volume portions ofmethylene chloride and then with two one-fourth by volume portions ofmethylene chloride. The combined methylene chloride extracts were washedwith two onetenth by volume portions of a two percent aqueous solutionof sodium bicarbonate and then with two one-tenth by volume portions ofwater. After drying the methylene chloride with about three to fivegrams of anhydrous sodium sulfate per liter of solvent and filtering,the solvent was removed by distillation. The semi-crystalline residueWeighing 4.988 grams was triturated four times, each time with sixmilliliters of ice cold methylene chloride. Remaining insoluble was 630milligrams of crystals melting at 194 to 199 degrees centigrade.Recrystallization of this with a mixture of three milliliters ofmethanol and ten milliliters of ether resulted in 404 milligrams ofcrystals melting at 205 to 210 degrees centigrade. Tworecrystallizations from the same solvents above gave 132 milligrams of1la,l7a,21trihydroxy-4-pregnene-3,20-dione having a constant meltingpoint of 209 to 2l2 de grees centigrade, and depending upon itscrystal'form, of 217 to 219 degrees centigrade, of plus 113 degrees(1.568 in methanol).

Analysis-Calculated for C H O C, 69.58; H,8.35. Found: C, 69.26; H,8.34,

PREPARATION 2 11a,17u,21-trihydrpxy-4-pregnene-3,20-dione In the samemanner as in Preparation 1, l7a-hydroxy-21-acetoxy-4-pregnene-3,20-dione is converted to 11oc,170c,21-trihydroxy-4-pregnene-3,20-dione by the action of Rhizopus nigricansor Rhizopus arrhizus.

PREPARATION 3 115,1 7m,21-trihydr0xy-4-pregnene-3,20-di0ne A medium wasprepared from 0.5 percent peptone, two percent dextrose, 0.5 percentsoybean'meal, 0.5 percent KH PO 0.5 percent sodium chloride and 0.3percent yeast extract in tap water. To 200 milliliters of thissterilized medium was added an inoculum of the vegetative mycelia ofCunninghamella blakesleeana. The spores had first been transferred froma spore slant to a broth medium and the broth medium was aerobicallyincubated at 24 degrees centigrade for 24 to 72 hours in a reciprocatingshaker until the development of vegetative growth. The inoculated mediumcontaining added vegetative mycelia of Cunninghamella blakesleeana wasincubated for 48 hours at 24 degrees centigrade following which wasadded 66 milligrams of compound S, 11-desoxy-l7-hydroxycorticosterone insolution in a minimum of ethanol and incubation was maintained for sevenhours at 24 degrees centigrade. The beer containing steroid was dilutedwith 800 milliliters of acetone, shaken one hour on a reciprocatingshaker and filtered. The cake was suspended in 500 milliliters ofacetone, shaken another hour and again filtered. The filtrates werecombined and the acetone was volatilized under reduced pressure at fiftydegrees centigrade. Actone was then added, if necessary, to bring theconcentration to twenty percent acetone and this resulting aqueousacetone solution was extracted five times each with one-third volume ofSkellysolve B petroleum ether to remove fatty materials. These extractswere back washed two times with one-tenth volume of twenty percentaqueous acetone and the washings were added to the main acetone extract.The combined acetone extracts were extracted six times with one-fourthvolumes of ethylene dichloride and the ethylene dichloride extract wasevaporated under vacuum to leave the steroid residue. This steroidresidue was taken up in a minimum of methylene chloride and applied tothe top of a column packed with thirty grams of silica which had beenpreviously triturated with 21 milliliters of ethylene glycol. Thenvarious developing mixtures, saturated with ethylene glycol, were passedover the column. Cuts were made as each steroid was eluted as determinedby the lowering of the absorption of light at 240 mu, in accordance withTable I on the automatic chromatographic fraction cutter.

TABLE I.-AUTOMATIC CHROMATOGRAPHIC FRAC- TION CUTTER A 7.7 milligramportion of band 5 was taken up in a minimum of acetone and refrigerateduntil crystals separated. This cold acetone mixture was centrifuged andthe supernatant liquid removed by pipette. To the remaining crystals, afew drops of ice-cold ether-acetone, three to one mixture, were added,shaken, recentrifuged and the supernatant wash liquid removed bypipette. The ether-acetone wash was repeated. The resulting crystalswere dried under vacuum yielding 3.3 milligrams of pure compound F,l7-hydroxycorticosterone also known as 1 1,8, 17a,21-trihydroxy-4-pregnene-3 ,20-dione.

Appreciable amounts of compound E, ll-dehyd-ro-l7-hydroxycorticosterone, cortisone, are also produced in the abovepreparation.

PREPARATION 4 11,8,17a,21-trihydr0xy-4-pregnerte-3,20-di0ne The sporesof Cunninghamella blakesleeana were transferred from an agar slant to abroth medium and aerobically incubated at 24 degrees centigrade for 24to 72 hours in a reciprocating shaker until the development ofvegetative growth.

Six hundred milliliters of this vegetative growth was used to inoculatetwelve liters of a sterile medium containing grams of dextrin, 480 gramsof corn steep solids, twelve grams of calcium carbonate and sixty gramsof sodium chloride which was made to twelve liters with tap water andadjusted to pH 6.5 with sodium hydroxide before sterilization. Theinoculated medium was. incubated at 26 degrees centigrade in a fivegallon bottle with stirring and aeration for 48 hours.

The seed thus prepared was added asceptically to 240 liters of a sterilemedium prepared from 0.5 percent soybean meal, two percent dextrose, 0.5percent d-n'ed brewers yeast, 0.5 percent sodium chloride, 0.5 percentKH PO made to volume with tap water and adjusted to pH 6.4. Fifty cubicfeet of air per hour was passed through the stirred medium for seventeenhours at which time the pH was 6.1. Then twelve grams of:,2l-dihydroxy-4-pregnene-3,20-dione in eight liters of redistilledethanol which had been sterilized by ultrafine sintered glass filtrationwas added and the incubation con tinued for 11.5 hours.

Four kilograms of diatomaceous earth was added to the beer, the mixturewas passed through a filter press and the cake then washed with fortyliters of tap water. The clear filtrate was extracted with three fiftyliter por- ,tions of ethylene dichloride. The extract was then eon- 2centrated under vacuum in a nitrogen atmosphere at fifty degreescentigrade to a volume of 600 milliliters.

Three hundreds grams of magnesium silicate was poured into acetonecontained in a chromatographic column 4.5 centimeters in diameter and 36centimeters long. The acetone was replaced with ethylene dichloride andthe column washed with two liters of ethylene dichloride. The'ethylenedichloride concentrate was added to the top of the column and developedwith ten liters of dry ethylene dichloride. The adsorbed steroids werethen eluted from the column with two liters of dry acetone. Afterremoval of the acetone at fifty degrees centigrade under nitrogen, theresidue was taken up in 200 milliliters of ethyl acetate and washedtwice with fifty milliliters of an equal volume mixture of a two percentaqueous sodium carbonate solution and a three percent aqueous sodiumbicarbonate solution. The extract was then washed with fifty milliliterportions of water until the washes were neutral. The combined waterwas-hes were b ack extracted with thirty milliliters of ethylenedichloride and this together with a further 100 milliliters of the samesolvent were added to the ethyl acetate solution of the steroids.Evaporation of the mixed solvents under anaerobic conditions at lowtemperature left a friable brown mass. The latter material was dissolvedin 100 milliliters of chloroform, chilled to four degrees centigrade.The crystals which formed were removed by filtration and dried in vacuumto give 403 grams of steroids containing 2.41 grams of11fi,17tt,21'trihydroxy-4- pregnene-3,20-dione and 1.19 grams ofcortisone.

The mother liquor, upon concentration, gave an additional 834 milligramsof crystals containing 164 milligrams of 11,8,17ot,21trihydroxy-4-pregnene-3,ZO-dione and 433 milligrams of cortisone.

EXAMPLE 1 21 -acetxy-1 1 0a,] 7ot-dihydroxy-4-pregnene-3,20-di0ne ASOO-milligram sample of 11a,17a,2l-trihydr0Xy-4- pregnene-3,20-dione wasdissolved in four milliliters of pyridine. To this solution 155milligrams of acetic anhydride was added dropwise. The reaction mixturewas kept at room temperature 24 hours and then diluted with seventymilliliters of ice cold water, followed by extraction twice with fiftymilliliters and twice with 25 milliliters of methylene dichloride. Theextract was washed with two 25 milliliter port-ions of five percenthydrochloric acid with 25 milliliters of ten percent sodium bicarbonatesolution, and with water to neutrality. The extract was dried withanhydrous sodium sulfate and the solvent evaporated. The oily residuewas dissolved in 125 milliliters of ethylene dichloride andchromatographed over eight grams of Florisil synthetic magnesiumsilicate. Solvents used to develop the column were in 100-milliliterportions in the following order: 1 and 2 ethylene dichloride, 3 to 5ethylene dichloride with acetone 25:1 ratio respectively by volume, 6and 7 ethylene dichloride with acetone 15:1 ratio, 8 to 11 ethylenedichloride with acetone 12:1 ratio, 12 to 14 ethylene dichloride withacetone :1 ratio, 15 to 18 ethylene dichloride with acetone 8:1 ratio,19 and 20 ethylene dichloride with acetone 5:1 ratio, 21 to 23 ethylenedichloride with acetone 2:1 ratio, 24 and 25 acetone.

Chromatographic fractions fifteen through twenty were combined, and thesolvent was then evaporated to yield 162 milligrams of21-acetoxy-11a,17u-dihydroxy-4-p-regnene-3,20-dione.

EXAMPLE 2 11a,21-diacet0xy-17a-hydr0xy-4-pregnene-3,20-di0ne Fractionalextracts, number eight to eleven inclusive, from the chromatographicdevelopment in Example 1 were freed of solvent, by evaporation, combinedand recrystallized from acetone by dropwise addition of petroleum ether.Three recrystallizations yielded milligrams of11tt,21-diacetoxy-17a-hydroxy-4-pregnene-3,20-

dione, melting at 198 to 202 degrees centigrade, [011 of plus degrees.(1.145 in chloroform), k equal to 33.33. ture.

Analysis-Calculated for C25H24O7I C, 67.24; H, 7.67. Found: C, 67.43; H,7.94.

EXAMPLE 3 21 -acetoxy-1 1,6,1 7a-dihydroxy-4-pregnene-3,20-di0ne To asolution of 500 milligrams of11,6,17a,21-trihydroXy-4-pregnene-3,20-dione in five milliliters ofpyridine, milligrams of acetic anhydride was added dropwise. After beingmaintained at room temperature for 24 hours, the reaction mixture wasdiluted with 75 milliliters of ice-cold water, and the diluted mixturewas extracted twice with fifty-milliliter portions of cold ether andtwice with 25-milliliter portions of methylene chloride. The combinedextracts were washed with two 25-milliliter portions of cold fivepercent hydrochloric acid, with 25 milliliters of two percent sodiumbicarbonate solution, and with water to neutrality. The washed extractwas dried over anhydrous sodium sulfate, filtered, and the solventevaporated. Recrystallization from acetone by the Infrared spectrumvertified the indicated strucdropwise addition of petroleum etherproduced 2l-acetoxy-l 1 8, 17u-dihydroxy-4-pregnene-3 ,20-dione.

EXAMPLE 4 21 -pr0pi0nyloxy-I 1 ot,1 7a-dihydr0xy-4-pregnene-3,20- dioneand 110:,21-dipr0pi0nyl0xy-4-pregnene-3,201di0ne Following the procedureof Example 1, using the equivalent proportion of propionic anhydride inplace of acetic.

anhydride and separating by chromatography produced 21 propionyloxy11a,17a-dihydroxy-4-pregnene3,20- dione and110:,21-dipropionyloxy-17a-hydroxy-4-pregnene-3,20-dione.

EXAMPLE 5 21-propi0nyl0xy-1 15,1 7a-dihydroxy-4-pregnene-3,20- dioneFollowing the procedure of Example 3, using the equivalent proportion ofpropionic anhydride in place of acetic anhydride, produced 21-propionyloXy-1 1B, 17a-dihydroxy- 4-pregene-3,20-dione.

EXAMPLE 6 21 -trimethylacet0xy-1l 0a,] 7ot-dihydr0xy-4-pregncue-3,20-dione and 11 ot,21-di( trimethylacetoxy -17a-hydr0xy- 4-pregn'ene-3,20-di0ne To one gram of 1lot,17a,21-trihydroxy-4-pregnene-3,

Following the procedure of Example 6, using the equivalent proportion offl-cyclopentylpropionyl chloride in place of trimethylacetyl chlorideproduced 21-,6-cyclopentylpropionyloxy 11ot,17u dihydroxy 4 pregnene-3,20-dione and1104,2l-difl-cyclopentylpropionyloxy-17mhydroXy-4-pregnene-3,2O-dione.

EXAMPLE 8 21 benzoxy 11,17a dihydroxy 4 pregrtene 3,20 dione and 11tt,21-dibenzoxy-17at-hydr0xy-412regnene- 3,20-di0ne Following theprocedure of Example 6, using the equivalent proportion of benzoylchloride in place of trimethylacetyl chloride produced21-benzoxy-1leaflet-dihydroxy- 4 pregnene -3,2() dione and 110;,21dibenzoxy 17ehydroxy-4-pregnene-3,20-dione.

Other mono-esters and di-esters of11e,l7a,21-trihydroxy-4-pregnene-3,20-dione and mono-esters of 115,17,21-trihydroxy-4-pregnene-3,20-dione are prepared according to variousacylation procedures such as illustrated in the examples, or by reactionwith ketene, ketenes of selected acids, selected acids, acid anhydrides,or acid chlorides, in an organic solvent such as pyridine or the like.Representative 21 acyloxy 11a,17a dihydroxy- 4 pregnene 3,20 diones,11a,2l diacyloxy 17ozhydroxy 4 pregnene 3,20 diones, and 21-acyloxy-116,17a dihydroxy 4 pregnene 3,20 diones thusprepared include one toeight carbon atom carboxylic acid acyloxy esters of saturated orunsaturated aliphatic, carbocyclic, cyclo-aliphatic, aryl, arylalkyl,alkaryl, mono, di or polycarboxylic acids which form ester groups suchas, for example, formyloxy, acetoxy, propionyloxy, dimethylacetoxy,trimethylacetoxy, butyryloxy, valeryloxy, hexanoyloxy, heptanoyloxy,actanoyloxy, benzoxy, phenylacetoxy, toluoyloxy, napthoyloxy,cyclopentylformyloxy, 8-cyclopentylpropionyloxy, acrylyloxy,cyclohexylformyloxy, the half and di-esters of malonic, maleic,succinic, glutaric and adipic acids, and the like. The acids may alsocontain non-interfering substituents, such as mono or poly halo, chloro,bromo, hydroxy, methoxy, and the like, if desired.

If a mixed ester involving two different acyl groups is desired, the11a,17a,21-trihydroxy-4-pregnene-3,ZO-dione may be partially esterifiedwith one acylating agent and the resulting mono-ester may then becompletely este-rified with an acylating agent which introduces adifferent acyl group. Thus1lot-propionyloxy-ZZl-fi-cyclopentylpropionyloxy-l7u-hydroxy-4-pregnene-3,ZO-dioneor other mixed esters of the herein mentioned acid groups may beprepared.

EXAMPLE 9 Cortisone acetate 162 milligrams of21-acetoxy-11a,17a-dihydroxy-4- pregnene-3,20-dione, obtained in Example1, was dissolved in ten milliliters of glacial acetic acid and oxidizedby adding dropwise a solution of thirty milligrams of chromum trioxide(CrO in 0.5 milliliter of water and two milliliters of acetic acid.After six hours at room temperature, the green solution was diluted withtwenty milliliters of methanol and evaporated under reduced pressure.The crystalline residue was suspended in 25 milliliters of ten percentsodium bicarbonate solution and extracted with four 25 milliliterportions of a mixture of four parts of ether to one part of chloroformby volume. The extract was washed thrice with water and dried overanhydrous sodium sulfate.

The resultant crystalline oxidation product, weighed 166 milligrams.Recrystallization from acetone yielded 138.5 milligrams of compound Eacetate, 17a-hydroxy- 21-acetoxy-4-pregnene-3,11,20-trione, otherwiseknown as cortisone acetate, with a melting point of 243 to 245 degreescentigrade, k equal to 36.33, [a] of plus 169 degrees (0.3657 inchloroform).

Analysis.-Calculated for C I-1 C, 68.61; H, 7.52. Found: C, 68.52; H,7.61.

In a similar manner, oxidizing the herein described 21 acyloxy :,170:dihydroxy 4 pregnene 3,20- diones or 21-acyloxy-ll19,17a-dihydroxy-4-pregnene-3,20- diones with, for example, chromiumtrioxide in acetic acid produces the corresponding acid esters ofcortisone, 21 acyloxy 17a hydroxy 4 pregnene 3,11,20 triones where inthe acyloxy group is the same as that in the starting 21 acyloxy 11,170;dihydroxy 4 pregnene- 3,20-diones.

It is to be understood that the invention is not to be limited to theexact details of operation or exact organisms and compounds shown anddescribed, as obvious modifications and equivalents will be apparent toone skilled in the art, and the invention is therefore to be limitedonly by the scope of the appended claims.

We claim:

1. A process for the production of an11,17a,21-trihydroxy-4-pregnene-3,20-dione ester comprising aerobicallycontacting 1711,21 dihydroxy 4 pregnene 3,20- dione with the oxygenatingactivity of a species of fungus of the order Mucorales to produce1l,l7a,21-trihydroxy- 4-pregnene-3,20-dione and react-ing thethus-produced 1l,17,21-trihydroxy-4-pregnene-3,ZO-dione with anacylating agent to produce an ester of 11,17a,21-trihydroxy-4-pregnene-3,20-dione.

2. The process of claim 1 wherein the acylating agent is an organiccarboxylic acid.

3. The process of claim 1 wherein the acylating agent is a carboxylicacid acyl halide.

4. The process of claim 1 wherein the acylating agent is aceticanhydride.

5. The process of claim 1 wherein the species of fungus is of the familyMucoraceae.

6. The process of claim 1 wherein the species of fungus is of the genusRhizopus.

7. The process of claim 1 wherein the species of fungus is of the genusCunninghamella.

8. The process of claim 1 wherein the fungus is Rhizopus nigricans,Rhizopus arrhizus, or Cunninghamella blakesleeana.

9. A process comprising aerobically contacting 17a,2l-dihydroxy-4-pregnene-3,20-dione with the oxygenating activity of aspecies of fungus of the order Mucorales to produce11,17a,21-trihydroxy-4-pregnene-3,20-dione, reacting the thus-produced11,17a,21-trihydroxy-4-pregnene-3,20-dione with an acylating agent toproduce a 21- acyloxy-11,17ot-dihydroxy-4-pregnene-3,20-dione andreacting the thus-produced 21-acyloxy-11,17a-dihydroxy4-pregnene-3,20-dione with an oxidizing agent to produce a2l-acyloxy-17ot-hydroxy-4-pregnene-3,11,20-trione.

10. The process of claim 9 wherein the species of fungus is of the genusRhizopus.

11. The process of claim 9 wherein the species of fungus is of the genusCunninghamella.

12. The process of claim 9 wherein the fungus is Rhizopus nigricans,Rhizopus arrhizus, or Cunninghamella blakesleeana.

13. The process of claim 9 wherein the acylating agent is aceticanhydride and the oxidizing agent is chromium trioxide in aceticanhydride.

References Cited in the file of this patent UNITED STATES PATENTS

1. A PROCESS FOR THE PRODUCTION OF AN11,17A,21-TRIHYDROXY-4-PREGNENE-3,20-DIONE ESTER COMPRISING AEROBICALLYCONTACTING 17A,21 - DIHYDROXY - 4 - PRGNENE - 3,20DIONE WITH THEOXYGENATING ACTIVITY OF A SPECIES OF FUNGUS OF THE ORDER MUCORALES TOPRODUCE 11,17A,21-TRIHYDROXY4-PREGNENE-3,20-DIONE AND REACTING THETHUS-PRODUCED 11,17A,21-TRIHYDROXY-4-PREGNENE-3,20-DIONE WITH ANACYLATING AGENT TO PRODUCE AN ESTER OF11,17A,21-TRIHYDROXY4-PREGNENE-3,20-DIONE.